Q. Wang et al.
EuropeanJournalofPharmaceuticalSciences118(2018)32–39
cularly suitable for tumor tissue imaging. What is more, the spir-
ocyclization of SiR is an attractive strategy for designing NIR (near
infrared) fluorescent probes to detect some ions by a reversible ring-
design principle, some SiR-based fluorescent dyes have been exploited
for imaging Hg2+, OH−1 and the proteins of interest from living cells.
As a promising recognition group, boronic acid participates in such an
equilibria: in a neutral or mildly basic environment, it binds with the
substances containing hydroxyl groups and forms the corresponding
boronate which can be reversibly hydrolyzed to boronic acid under
designed and synthesized a SiR-based spirocyclic derivative SiRB (Si-
rhodamine and boronic acid), in which a boronic acid group was in-
anhydrous Na2SO4. After removal of the solvent under reduced
pressure, the residue was purified by silica gel column
chromatography with petroleum ether/dichloromethane (2/1, v/v) as
eluent. The pure compound 1 was a white solid (yield: 71%).
2.2.1.2. Synthesis of SiX (Si-xanthone). The compound 1 (5 mmol) and
anhydrous THF (tetrahydrofuran) (20 mL) were added to a pre-dried
flask flushed with nitrogen. The solution was cooled to −78 °C, 1.3 M s-
BuLi (sec-butyllithium) (10.7 mL) was added, and the mixture was
stirred for 30 min. At the same temperature, a solution of SiMe2Cl2
(10 mmol) in anhydrous THF (10 mL) was slowly added, and the
mixture was slowly warmed to room temperature, then stirred for
3 h. The reaction was quenched by the addition of 2 M hydrochloric
acid. Then the mixture was neutralized with NaHCO3 solution, and
extracted with CH2Cl2. The organic layer was washed with brine and
dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure and the crude was used without further purification. KMnO4
solution (15 mmol) was added to the crude dissolved 50 mL acetone at
0 °C in small portions over a period of 1 h with vigorous stirring. The
mixture was stirred for another 1 h at the same temperature, then
diluted with CH2Cl2 (50 mL), filtered through paper filter and
evaporated to dryness. The residue was purified by column
chromatography on silica gel to give pure SiX as a yellow solid
(yield: 64%).
troduced as a pH-sensitive group. SiRB displayed the reversible H+
-
triggered ring-opening process of the corresponding spirocyclic struc-
tures accompanied by the remarkable chromogenic and fluorogenic
changes. Furthermore, SiRB probe showed the prominent fluorescence
changes in the pH range from 7.6 to 5.8, demonstrating an ideal suit-
ability for specific labeling of acidic microenvironment of tumor and
tracking the pH changes.
As a biodegradable polymer organic compound, PLGA (poly lactic-
co-glycolic acid) has good biocompatibility, non-toxicity, good en-
capsulation and film-forming properties. And it is widely used in
pharmaceutical, medical engineering materials and modern industry as
is fabricated for ratiometric fluorescence imaging and pH detection. It is
found that two types of NIR dyes, SiRB and ICG (indocyanine green),
can be effectively encapsulated into PLGA via hydrophobic interaction
and induce the self-assembly of PLGA to form polymer-dye nano-
particles, with ICG, whose absorbance and fluorescence are inert to pH
et al., 2014). The pH-responsive dye SiRB could act as a pH indicator
under ratiometric fluorescence imaging. The accurate detection of
tumor pH by fluorescence imaging was realized after intravenous in-
jection of this nanoparticle. The gradual acidification of the tumor
microenvironment during the tumor growth, as well as the instant
tumor pH changes upon injection of external buffers, was vividly ob-
served by this method.
2.2.1.3. Synthesis of Br-B(2-(2′-bromophenyl)-6-butyl dioxazaborocan). N-
butyldiethanolamine (20 mmol) was added to
a suspension of 2-
bromophenylboronic acid (20 mmol) in anhydrous toluene (30 mL). The
mixture was heated at 50 °C for 2 h. After cooling to room temperature, the
toluene was evaporated under reduced pressure. The remaining clear
colorless crude oil was treated with heptane to remove the residual
toluene. The resulting suspension was allowed to stand at room
temperature overnight. The precipitated solid was collected by filtration,
washed with heptane, and dried overnight to give the title compound Br-B
as a white solid (yield: 58%).
2.2.1.4. Synthesis of SiRB. To a dried flask flushed with nitrogen,
compound Br-B (1.0 mmol) and anhydrous THF (10 mL) were added.
The solution was cooled to −78 °C, 1.6 M n-BuLi (n-Butyllithium)
(0.75 mL) was added over a period of 10 min and the mixture was
stirred for further 20 min. At the same temperature, a solution of SiX
(1.0 mmol) in anhydrous THF (10 mL) was slowly added. The mixture
was allowed to stir for 20 min at −78 °C, then allowed to warm to room
temperature gradually. After stirring for further 1 h, 6 M HCl solution
(10 mL) was added and the mixture was stirred for an additional
30 min. The resulting blue solution was neutralized with NaHCO3
solution, and extracted with CH2Cl2. The organic layer was washed
with brine and dried over anhydrous Na2SO4. After removal of the
solvent under reduced pressure, the residue was purified by column
chromatography on silica gel to give SiRB as a light blue solid (yield:
52%).
2. Materials and methods
2.1. Materials
3-Bromo-N,N-dimethylaniline, 2-bromophenylboronic acid (97%),
N-butyldi ethanolamine (98%) and indocyanine green (ICG) were
purchased from Aladdin. poly lactic-co-glycolic acid (PLGA, 99%) was
obtained from Fu Zhong Pharmaceutical Technology Co., Ltd (Beijing,
China). N-Butyl lithium, sec-butyllithium, heptane were obtained from
Sigma-Aldrich (St. Louis, Missouri, USA). Tetrahydrofuran (THF), me-
thylene chloride and acetic acid were purchased from Fuyu Fine
Chemical Co., Ltd (Tianjin, China). Other reagents were acquired from
Sigma-Aldrich. All the reagents were of analytical grade and used
without further purification.
2.2.2. Synthesis of PLGA-ICG-SiRB
The PLGA-ICG-SiRB nanocomposites were obtained by self-as-
sembly and subsequently loaded into PLGA nanoparticles. Briefly, 1 mg
synthesized SiRB, 0.2 mg ICG and 50 mg PLGA were firstly dispersed in
1 mL acetone. The mixture was added to 40 mg BSA (bovine serum
albumin) dispersed in 4 mL deionized water, and stirred overnight in
the dark. PLGA-ICG-SiRB nanoparticles were obtained after purification
of the suspensions by 8000–12,000 D dialysis bag.
2.2. Methods
2.2.1. Synthesis of SiRB
2.2.1.1. Synthesis of 4,4′-methlenebis (3-bromo-N,N-dimethylaniline)
(compound 1). A solution of 3-bromo-N,N-dimethylaniline (25 mmol)
in AcOH (80 mL) was added to 37% formaldehyde (125 mmol), and the
mixture was stirred at 85 °C for 90 min. After cooling to room
temperature, the reaction mixture was carefully neutralized with
saturated aqueous NaHCO3 and extracted with CH2Cl2 for three
times. The organic layer was washed with brine and dried over
2.2.3. Characterization of PLGA-ICG-SiRB
The PLGA-ICG-SiRB nanoparticles were characterized by transmis-
sion electron microscopy (TEM, Tecnai G2 20, FEI, USA). UV–vis ab-
sorption spectra were recorded using a UV–vis spectrometer (Lambda
35, Perkinelmer, USA). Fluorescence spectra were recorded with a RF-
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